It has been known that the quantity of steroid hormones and/or metabolites thereof contained in human body fluid or excreted fluid is correlated with physiological and pathological states of human beings. Therefore quantitative analysis of steroid hormones and/or metabolites thereof is useful for purposes of diagnosis and clinical examination.
However, the quantity of steroid hormones and metabolites thereof contained in human body fluid or excreted fluid is generally small, and the variation in quantity of steroid hormones and metabolites thereof due to a specific physiological or pathological states is also small.
Consequently quantitative analysis of steroid hormones and/or metabolites thereof requires extremely high accuracy.
Recent developments in analytical instruments make it possible to conduct extremely accurate quantitative analysis. Such instruments are highly elaborate, and installation and maintainance of such instruments involve high costs in general. Furthermore, manipulation of such instruments is not easy for persons who are not skilled, and often requires troublesome and time-consuming preliminary treatment of samples to be analyzed.
Therefore it is significant to provide a means for enabling accurate and rapid quantitative detection of steroid hormones and/or metabolites thereof without necessitating special training and special instruments.
It has been known that quantitative detection of steroids can be accomplished by immunological methods. One of the typical immunological methods utilizes the specificity of the antigen-antibody reaction and visibility of the agglutination and agglutination inhibition reaction thereof. More specifically, in the typical prior art, the type of steroid to be detected is bonded to protein to give a steroid-protein conjugate, then a reagent is prepared having latex particles sensitized with the conjugate and antiserum is prepared by injecting the conjugate into a mammal. The thus obtained antiserum may react with the steroid to be detected and also may react with the reagent. Therefore if a given quantity of a test sample of human body fluid or excreted fluid is collected and mixed with a given quantity of said antiserum, a reaction takes place therebetween. Then a given quantity of the reagent is added to the liquid mixture of the test sample and the antiserum. If an excess amount of antiserum remains in the fluid mixture of said test sample and the antiserum, antigen-antibody reaction between the remaining antiserum and the reagent can be observed as an agglutination reaction, and if the antiserum is just neutralized with the steroid contained in the test sample or an excess amount of steroid remains in the fluid mixture no antigen-antibody reaction occurs, and this can be observed as and agglutination inhibition reaction. Therefore, if serially diluted test samples are tested with the antiserum, which is used at a predetermined titer, and the reagent, quantitative assay of the steroid and/or metabolites thereof can be performed.
One of such prior art methods is disclosed in Japanese Early Opened Patent application Publication No. 51-112513 (Filing Date: Mar. 25, 1975; Inventor: Osamu Kanemitsu; Applicant: Asahi Kasei Kogyo Kabushiki Kaisha; Date of Laying-Open: Oct. 5, 1976). This first prior art publication refers to a method for immunological quantitative assay of dehydroepiandrosterone sulphate (which is an intermediate in the biosynthesis of a sex hormone), but this prior art adds nothing new to the aforementioned typical prior art. Though these methods of the prior art provide useful diagnostic testing methods, it has been strongly desired to increase the sensitivity of such immunological quantitative assay, since this makes it possible to detect slighter variations of steroid quantity and to provide increased accuracy for conventional use.
Meanwhile, human body fluid or excreted fluid usually contains many kinds of compounds besides steroids, and the amounts of these compounds are usually far greater than the steroid content. Some of these compounds, for example, protein and saccharide, affect immunological assay, and it is necessary to remove them or dilute the body fluid or excreted fluid to be tested to the extent where no interference is caused. Removal of the interfering compounds requires a complicated process, and in clinical analysis which requires simplicity and rapidity, it is necessary to dilute the fluid to be tested. This makes it necessary to increase the sensitivity of such immunological assay.
Adjustment of sensitivity is referred to in the Japanese Early Opened Patent application Publication No. 50-123819 (Filing Date: Mar. 14, 1974; Inventor: Hideaki Manita et al; Applicant: Teikoku Zoki Seiyaku Kabushiki Kaisha; Date of Laying-Open: Sept. 29, 1975).
This second prior art publication discloses a method for immunological assay of steroids contained in human body fluid or excreted fluid which utilizes an antibody obtainable from a mammal immunized with steroid conjugated with antigenic protein having free amino group and sensitized carriers wherein carriers are sensitized with the steroid conjugated with protein which is different from the antigenic protein. In this second prior art method, adjustment of sensitivity can be achieved by varying the amount of the steroid-protein conjugate for sensitizing latex particles in the reagent and by varying the concentration of the antiserum. Though the descriptions in the second prior art publication are not necessarily clear, even if they may suggest that a smaller amount of excess antiserum can agglutinate sensitized latex particles when the latex particles are sensitized with a smaller amount of the steroid-protein conjugate, this approach may not give a significant improvement of sensitivity for the reason mentioned below. Originally, latex particles have a tendency to show non-specific agglutination when a proper stabilizing agent is not added thereto, and this non-specific agglutination cannot be distinguished from specific agglutination due to the antigen-antibody reaction. The steroid-protein conjugate used for sensitization of latex particles may act as a stabilizing agent for eliminating this non-specific agglutination of the latex particles proper. Accordingly the adjustment of sensitivity in the second prior art method is limited to a certain extent where non-specific agglutination is eliminated, and it is not possible to increase sensitivity beyond the limitation.
An approach to overcoming this limitation is considered to use the techniques disclosed in Japanese Patent Publication No. 49-11407 (Filing Date: Dec. 29, 1970; Inventor: Tadamitsu Sudo; Applicant: Teikoku Zoki Seiyaku Kabushiki Kaisha; Publication Date: Mar. 16, 1974) and Japanese Early Opened Patent application Publication No. 50-82230 (Filing Date: Nov. 29, 1973; Inventor: Tadamitsu Sudo et al; Applicant: Teikoku Zoki Seiyaku Kabushiki Kaisha; Date of Laying-Open: July 3, 1975). In these third and fourth prior art publications, it is disclosed that latex particles can be stabilized by making latex particles absorb an immunologically inert protein before or after the latex particles are sensitized with steroid-protein conjugate or antibody. Combining the second prior art publication and the third or fourth prior art publication, there is a possibility of obtaining sensitized latex particles having increased sensitivity with the latex particles being sensitized with a smaller amount of steroid-protein conjugate and being stabilized with an immunologically inert protein. It has been found, however, that this stabilization of latex particles by the inert protein tends to affect the immunologically specific agglutination.
Japanese Early Opened Patent application Publication No. 48-49918 (Filing Date: Oct. 26, 1972; Inventor: John Anthony Coppola et al; Applicant: American Cyanamid Company; Date of Laying-Open: July 14, 1973) discloses a method for preparation of antibody which has excellent specificity against progesterone and applicability of the antibody for radioimmunoassay and an agglutination test for determination of the concentration of progesterone in test samples. However, this fifth prior art publication does not generally refer to improvement of sensitivity for detection of a variety of steroids.
Moreover, in this fifth prior art publication, it is apparently stated that the steroid (hydroxyprogesterone)-protein conjugate has 15-40 molecules of steroid per molecule of protein, and this conjugate is used not only for preparation of antibody but also for sensitization of latex particles. Also, in the second prior art publication (Japanese Early Opened Patent application Publication No. 50-123819), estriol-16.alpha.-glucuronide--rabbit serum albumin conjugate is used for sensitization of latex particles, and the bonding ratio of steroid glucuronide to protein ranges from 27 to 30 moles per mole of protein used.
As will be seen from the foregoing, steroid-protein conjugates in which a relatively large number of molecules of steroid are bonded per molecule of protein have been used for sensitization of latex particles in the prior art (hereinafter the number of molecules of steroid which are bonded to a molecule of protein is simply referred to as the steroid bonding number).
The inventors have considered that further improvement of sensitivity would not be expected through conventional approaches, and the inventors' attempts have been concentrated on improvement of the conjugate proper. As a result, the inventors have found that when a steroid-serum albumin conjugate is prepared so that the steroid bonding number falls within a range of 0.5-7.0, and then latex particles are sensitized with such conjugate, the thus obtained sensitized latex particles show extremely high sensitivity, and that this is applicable irrespective of the sort of steroid and serum albumin used. As far as the inventors know, there is no reference which refers to decreasing steroid bonding number for increasing sensitivity of steroid detection.